Novel alkylphospholipid-DTC hybrids as promising agents against endocrine related cancers acting via modulation of Akt-pathway

Article abstract of DOI:10.1016/j.ejmech.2014.08.028

A new series of 2-(alkoxy(hydroxy)phosphoryloxy)ethyl dialkylcarbodithioate derivatives was synthesized and evaluated against endocrine related cancers, acting via modulation of Akt-pathway. Eighteen compounds were active at 7.24-100 μM against MDA-MB-231 or MCF-7 cell lines of breast cancer. Three compounds (14, 18 and 22) were active against MCF-7 cells at IC₅₀ significantly better than miltefosine and most of the compounds were less toxic towards non-cancer cell lines, HEK-293. On the other hand, twelve compounds exhibited cell growth inhibiting activity against prostate cancer cell lines, either PC-3 or DU-145 at 14.69-95.20 μM. While nine of these were active against both cell lines. The most promising compounds 14 and 18 were about two and five fold more active than miltefosine against DU-145 and MCF-7 cell lines respectively and significantly down regulated phospho-Akt. Possibly anti-cancer and pro-apoptotic activity was mostly due to blockade of Akt-pathway.

Full text of DOI:10.1016/j.ejmech.2014.08.028

European Journal of Medicinal Chemistry 85 (2014) 638e647
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Novel alkylphospholipid-DTC hybrids as promising agents against
endocrine related cancers acting via modulation of Akt-pathway*
,
Santosh Jangir a, Veenu Bala a c, Nand Lal a, Lalit Kumar a, Amit Sarswat a, Amit Kumar b,
Hamidullah b, Karan S. Saini b, Vikas Sharma b, Vikas Verma b, Jagdamba P. Maikhuri b,
, c, *
Rituraj Konwar b, Gopal Gupta b, Vishnu L. Sharma a
a Medicinal & Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
b Endocrinology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
c Academy of Scientific & Innovative Research, New Delhi 110001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of 2-(alkoxy(hydroxy)phosphoryloxy)ethyl dialkylcarbodithioate derivatives was synthe-
sized and evaluated against endocrine related cancers, acting via modulation of Akt-pathway. Eighteen
compounds were active at 7.24e100 mM against MDA-MB-231 or MCF-7 cell lines of breast cancer. Three
compounds (14, 18 and 22) were active against MCF-7 cells at IC50 significantly better than miltefosine
and most of the compounds were less toxic towards non-cancer cell lines, HEK-293. On the other hand,
twelve compounds exhibited cell growth inhibiting activity against prostate cancer cell lines, either PC-3
Received 15 February 2014
Received in revised form
7 August 2014
Accepted 7 August 2014
Available online 8 August 2014
or DU-145 at 14.69e95.20
mM. While nine of these were active against both cell lines. The most
Keywords:
promising compounds 14 and 18 were about two and five fold more active than miltefosine against DU-
145 and MCF-7 cell lines respectively and significantly down regulated phospho-Akt. Possibly anti-cancer
and pro-apoptotic activity was mostly due to blockade of Akt-pathway.
Alkylphospholipid
Dithiocarbamate
Akt-pathway
© 2014 Elsevier Masson SAS. All rights reserved.
Endocrine cancer
Miltefosine
Anti-proliferative agent
1. Introduction
being in the two endocrine related cancerseprostate cancer in men
(42%) and breast cancer in women (32%). These revelations have
Cancer is a group of diseases characterized by uncontrolled cell
proliferation and spread of malignant cells. About 77% of all cancers
are diagnosed in older persons with age 55 years or more. In US, the
lifetime risk from cancer is about 1 in every 3 person [1]. The Na-
tional Institute of Health (NIH) estimates that the overall costs of
cancer in 2007 were US $ 226.8 billion: $103.8 for direct medical
cost and $ 123.0 for indirect mortality costs [1]. The projections [2]
for cancer survivors and cost of care during 2010e2020 reveal that
there will be 18.1 million survivors in 2020 with the medical costs
to be increased by 27% from 2010. The largest medical cost increase
prompted the authors to design, synthesize and biologically eval-
uate chemical entities that may be useful in the management of
both prostate and breast cancer.
Recently the concept of hybrid molecules has been attracting a
lot of attention [3e6] as the presence of more than one active
pharmacophore in a single molecule leads to an entirely novel class
of compounds with better biological activity profile. It is supposed
that a single chemical entity can modulate multiple targets
simultaneously.
Alkylphosphocholines (APCs) [7] belong to a class of lipid mol-
ecules that include miltefosine (Fig. 1), perifosine (Fig. 1), erufosine
(Fig. 1) and edelfosine (Fig. 1), which have known anti-tumor
properties [8,9]. Unlike conventional chemotherapeutics, these
compounds structurally resemble membrane lipid components,
and thus, exhibit adequate drug delivery to target tumor tissues [9].
However, they can also target primarily the membrane-bound re-
ceptors associated with cancer-specific cellular phenotypes, such
as, cellular proliferation and survival. More specifically, the possible
mechanisms underlying the actions of APCs have been reported to
be the involvement of PI3K/Akt (phosphatidylinositide 3-kinase/v-
Abbreviations: APCs, alkylphosphocholines; DDTC, diethyldithiocarbamate;
HEK, human embryonic kidney; PI, propidium iodide; MMP, mitochondrial mem-
brane potential; ATCC, American type of cell culture collection; DMEM, Dulbecco
modified eagle medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-
lium bromide; PI3K, phosphatidylinositide 3-kinase.
*
C.D.R.I. Communication No. 210/2013/VLS.
* Corresponding author. Medicinal & Process Chemistry Division, CSIR-Central
Drug Research Institute, Lucknow 226031, India.
E-mail addresses: vlscdri1@rediffmail.com, vlscdri@gmail.com (V.L. Sharma).
http://dx.doi.org/10.1016/j.ejmech.2014.08.028
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

S. Jangir et al. / European Journal of Medicinal Chemistry 85 (2014) 638e647
639
to Scheme 1. Bromoethanol (1) was treated with phosphorus oxy-
chloride (2) to give 2-bromoethyl phosphorodichloridate (3). Sub-
sequent reaction of 3 with suitable alcohol furnished 2-bromoethyl
alkyl hydrogen phosphate (4e8), which after treatment with so-
dium salt of carbodithioic acid (9e12) provided 2-(alkoxy(hydroxy)
phosphoryloxy)ethyl dialkylcarbodithioate (13e32).
3. Biological evaluation
3.1. Cell inhibitory activity in breast and prostate cancer cells
The above compounds 13e32 were evaluated against breast
cancer cell lines, MCF-7 (estrogen responsive proliferative breast
cancer model), MDA-MB-231 (estrogen independent aggressive
breast cancer model); and prostate cancer cell lines, PC-3 (estrogen
responsive both
a and b), DU-145 (estrogen responsive b only) and
non-cancer cell line, HEK-293 (human embryonic kidney) using
MTT assay to assess cell inhibition [15,16]. Miltefosine, an alkyl-
phosphocholine was used as standard drug. The results (Table 1)
revealed that almost all the compounds except compounds 15 and
16 were inhibited both or either of MDA-MB-231 and MCF-7 breast
Fig. 1. Structures of known alkylphosphocholine, dithiocarbamate and envisaged
structure.
cancer cell lines. Standard drug, miltefosine had IC50 2.075
mM and
34.70 M against MDA-MB-231 and MCF-7 cells respectively. Out of
m
20 compounds, three compounds (14, 18 and 22) were active
against MCF-7 cells with IC50 better than miltefosine. Most of the
compounds were found to be less toxic towards non-cancer cell
lines, HEK-293, except compound 21, 31 and 32 which exhibited
some degree of toxicity. Compound 18 exhibited potent cell growth
inhibition against both MCF-7 and MD-MB-231 cells with IC50 of
Akt murine thymoma viral oncogene homolog) inhibition, their
downstream signaling pathways, and the induction of apoptosis
[7]. It is also known that APCs do not target the DNA, and they have
cell selective effects of inhibiting the proliferation of cancer cells at
low concentrations without affecting the normal cells [7]. Milte-
fosine (Fig. 1) shows a wide range of anti-tumor effects and as
Milterx®, it has been approved for the topical treatment of skin
metastases from breast cancer [7].
Likewise, dithiocarbamate is also a versatile pharmacophore
exhibiting various biological activities due to its sulfur based metal
chelating properties [10]. Diethyldithiocarbamate (DDTC; Fig. 1) is
capable of binding accumulated copper and forming a new complex
that could potently inhibit the proteasomal chemotrypsin-like ac-
tivity, decrease expression of androgen receptor (AR), estrogen
7.24 2.15 and 24.57 5.69 mM respectively. It may be inferred that
compound 18 is most promising compound with significant anti-
proliferative activity against breast cancer cell lines. On the other
hand twelve compounds (13e21, 23, 27 and 31) exhibited cell
growth inhibiting activity against prostate cancer cell lines, either
PC-3 or DU-145, at 14.69e95.20
23) were active against both the cell lines. The standard miltefosine
had IC50 of 19.90 M and 25.78 M against PC-3 and DU-145 cell
mM while nine of these (13e20 and
m
m
lines respectively. Compounds 14, 15 and 19 exhibited better ac-
tivity than miltefosine against DU-145 cell lines. Compound 14 and
15 were most promising as these two had lower IC50 values against
both the prostate cancer cell lines. Sodium piperidine-1-
carbodithioate was also evaluated against breast cancer cell lines,
MCF-7, MDA-MB-231 and prostate cancer cell lines, PC-3, DU-145
to assess the effect of hybridization of alkylphospholipids and DTC.
It has no effect against both the breast cancer cell lines and mildly
inhibited the growth of prostate cancer cell lines, PC-3 and DU-145
receptor (ER)-a and ER-b receptors proteins, and induce apoptosis
in both prostate and breast cancer cells [10]. Pyrrolobenzodiaze-
pine derivatives with DTC side chains inhibit the growth of prostate
and breast cancer cell lines [11]. Tetraethylthiuram disulfide
(Disulfiram, Fig. 1) and dithiocarbamate anion (Fig. 1) strongly
inhibited the proliferation of cancer cells of a variety of cell types
[12] by inhibiting the maturation of P-glycoprotein pump, an ATP-
driven 170-kd efflux pump on the plasma membrane that pumps a
variety of cytotoxic drugs out of the cell [13]. Another hybrid
molecule of butenolide and dithiocarbamate have anti-tumor
properties against several human cancer cell lines including the
prostate and breast cancer cells [14].
at IC50 25.5 1.25 mM and 18.7 0.84 mM respectively. These results
Thus, it was thought worthwhile to incorporate dithiocarba-
mate moiety into alkylphospholipid scaffold in place of amine
moiety (part of choline moeity) keeping the rest of the structure
same. The hybrid structures (envisaged structure, Fig. 1) though not
entirely alkylphosphocholine since it was modified in order to add
the alkyldithiocarbamate moiety, were therefore addressed as
alkylphospholipid. It was envisaged to arrive at a novel scaffold
(Fig. 1) having anti-proliferative activity against both the breast and
prostate cancer cell lines. Synthesis, biological activity and struc-
ture activity relationship (SAR) are being communicated.
2. Chemistry
The
2-(alkoxy(hydroxy)phosphoryloxy)ethyl
dia-
Scheme 1. Reagent and conditions; (a) CCl4, reflux, 10 h; (b) (1) ROH, CCl4, reflux, 10 h;
(2) H2O,rt, 6 h; (c) MeOH, reflux, 3 h.
lkylcarbodithioate derivatives (13e32) were synthesized according

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S. Jangir et al. / European Journal of Medicinal Chemistry 85 (2014) 638e647
Table 1
Cell inhibiting activity of the compounds (13e32).
Compd
Mean inhibition of cell proliferation (in terms of IC50 SEM in mM)
Anti-breast cancer activity
Cytotoxicity
Anti-prostate cancer activity
eNR1R2
eR3
MDA-MB-231
MCF-7
HEK-293
PC-3 cells
DU-145
13
14
15
eCH2(CH2)14CH3
eCH2(CH2)14CH3
eCH2(CH2)14CH3
69 2.88
~100 5.98
>100
72 8.39
29 1.37
>100
>100
>100
>100
65.7 1.1
34.3 1.4
32.1 0.3
28.88 4.8
14.69 1.0
17.6 1.4
16
17
eCH2(CH2)14CH3
eCH2(CH2)8CH3
>100
>100
>100
95.2 5.3
56.1 6.8
49.3 4.8
67 4.72
70 8.34
>100 16.2
46.14 3.0
18
19
eCH2(CH2)8CH3
eCH2(CH2)8CH3
24.57 5.69
11.4 3.72
7.24 2.15
70.7 1.11
70 1.71
62.5 2.5
32.9 2.9
29.27 2.9
23.95 1.5
43.3 5.07
20
21
22
23
eCH2(CH2)8CH3
eCH2(CH2)6CH3
eCH2(CH2)6CH3
eCH2(CH2)6CH3
29 6.6
99.6 3.65
53 4.37
26 4.39
78 7.56
>100 1.005
28.3 3.24
69 8.79
80 4.8
>100
82.3 8.4
47.1 2.9
>100
69 2.39
11.9 5.87
27 1.35
>100
>100 5.36
56.8 3.7
59.1 4.8
24
25
26
27
eCH2(CH2)6CH3
eCH2(CH2)4CH3
eCH2(CH2)4CH3
eCH2(CH2)4CH3
58.46 3.98
54 7.13
46 5.05
~100 6.99
62 6.57
~55
86 3.96
>100 4.75
>100
>100
>100
>100
>100
>100
>100
85 5.74
>100
17.19 5.00
70.5 6.31
77.4 8.4
28
eCH2(CH2)4CH3
73 6.13
58 4.96
>100
>100
>100
29
30
eCH2CH2CH3
eCH2CH2CH3
67 2.38
68 7.31
73 5.27
>100
>100
>100
>100
>100
>100
>100
31
eCH2CH2CH3
eCH2CH2CH3
38 2.98
74 6.38
54 6.67
>100
86.1 5.6
32
62 2.2
59 6.09
39.6 4.68
>100
>100
Miltefosine
2.075 0.12
34.7 11.7
e
19.9 0.7
25.78 1.4
Bold represents the values of most active compounds as compared to standard.
demonstrated that dithiocarbamate moiety of most active lead
compounds 14 and 18 alone does not show significant anti-cancer
activity.
Structureeactivity relationship (SAR) for breast cancer was
discussed according to the MCF-7 cell line as it is estrogen
responsive. Likewise activity against DU-145 cells was chosen for
SAR in prostate cancer as it is responsive to estrogen receptor
present in normal and malignant prostate. The SAR study estab-
lished that both ring size of amine and length of alkyl chain
significantly affected the activity. The IC50 values suggested that an
b

S. Jangir et al. / European Journal of Medicinal Chemistry 85 (2014) 638e647
641
increase in alkyl chain from propyl (29e32) to decyl (17e20)
increased from ~2% to ~9%, ~11% and ~13% on treatment with 5
10 M and 15 M of compound 14 (Fig. 3B).
mM,
enhanced the activity against MCF-7 cells while a further increase
to hexadecyl (13e16) resulted in decrease of activity. Whereas
against DU-145 cells the anti-proliferative activity increased with
length of alkyl chain and most active compound 14 had sixteen
carbon alkyl group. Thus, a ten carbon alkyl chain was most
appropriate for anti-breast cancer activity while sixteen carbon
chain was most desired against prostate cancer. The data further
revealed that an increase in ring size of amine from 5-membered
(13, 17, 21 and 25) to 6 (14, 18, 22 and 26) enhanced the activity
against MCF-7 cells for six to sixteen carbon alkyl chain while with
three carbon chain (29, 30) the activity reduced. Against DU-145
cells the activity enhanced on increasing the ring size from five
(13, 17) to six (14, 18) for long alkyl chains hexadecyl and decyl
whereas lower chains were less desirable. A further increase in ring
size of amine (15, 19, 23, 27 and 31) was not beneficial irrespective
of alkyl chain length and an additional nitrogen in amine residue
(16, 20, 24, 28 and 32) did not provide a better activity. The struc-
ture of most promising compounds 14 against DU-145 and 18
against MCF-7 cell lines (Fig. 2) suggested that a six membered
amine at dithiocarbamate residue was most appropriate for both of
these cell lines while optimum alkyl chain length was sixteen and
ten respectively.
m
m
3.4. Effect of compound 14 and 18 on expression of cell cycle and
apoptosis-related genes
The cell cycle phase distribution analysis results showed that
compound 18 induced G0/G1 arrest along with increase in sub-
diploid population in MCF-7 cells (Fig. 2A). Cell cycle progression
is a well-regulated process and dependent on various cyclins like
cyclins A1 and A2. In order to know the effect of compound 18 in
cell cycle and apoptosis-related genes which may be responsible for
cell cycle arrest, gene expression analysis [19] was carried out in
MCF-7 (Fig. 4A). The qPCR data showed that compound 18 treat-
ment resulted in down-regulation of expression of cyclins A1 and
A2 and anti-apoptotic gene, bcl-2. Likewise, increase expression of
Bax and decreased expression of Bcl-2 was observed in DU-145
cells treated with compound 14 as evident from western blot
result (Fig. 4B). A very significant increase in Bax/Bcl-2 ratio
(P < 0.001) was evident at 15 mM concentration (Fig. 4B').
3.5. Effect of compound 14 and 18 on mitochondrial membrane
potential (MMP)
It is interesting to observe that ten compounds (13, 14, 17e21,
23, 27 and 31) were active against both the breast and prostate
cancer cell lines. The most promising compounds 14 and 18 were
about two and five fold more active than miltefosine against DU-
145 and MCF-7 cell lines respectively and almost equally active
against MCF-7 and DU-145 cells. Therefore, it was thought worth-
while to study the mechanism of anti-proliferative action of com-
pounds 14 and 18 in respective cancer cell lines.
The membrane-permeant JC-1 dye is widely used as an indica-
tor of MMP. JC-1 dye exhibits potential-dependent accumulation in
mitochondria, indicated by a fluorescence emission shift from
green (~529 nm) to red (~590 nm). Consequently, mitochondrial
depolarization is indicated by a decrease in the red/green fluores-
cence intensity ratio [19]. The mitochondrial membrane potential-
sensitive color shift is due to concentration-dependent formation
of red fluorescent J-aggregates. When MCF-7 cells were treated
with compound 18 for 24 h, a significant increase in green fluo-
rescence was observed, suggesting loss of MMP in comparison to
untreated control (Fig. 5A). Similarly, a dose dependent increase
green fluorescence was also observed in DU-145 cells treated with
compound 14 (Fig. 5B).
3.2. Effect of compound 14 and 18 on cell cycle progression
Evasion of cell cycle arrest and apoptosis or combination of both
processes, is central to uncontrolled cell proliferation in cancer.
Therefore, candidate molecule capable of inducing cell cycle arrest
in cancer cells is considered to possibly have beneficial effect in
cancer. In order to investigate effect of lead compounds 14 and 18
on cell cycle phases, we conducted propidium iodide (PI)-staining
based cell cycle analysis as described in experimental section [17].
Our result showed that compound 18, a novel miltofosine-like
alkylphospholipid derivative could arrest the growth of MCF-7
cell line at G0/G1 phase (Fig. 2A). Cell cycle analysis by PI-
3.6. Effect of lead compound 14 and 18 on Akt phosphorylation
It is well-established that alkylphospholipid derivatives like
hexadecylphosphocholines miltefosine [20], perifosine [21] and
erufosine [22] downregulate Akt phosphorylation thereby blocking
Akt survival pathway. Since compounds (14 and 18) are alkyl-
phospholipid derivatives and share considerable homology with
the other pharmacologically active compounds of this class, the
effect of compound 14 and 18 was tested on phosphorylation of Akt
as described in experimental section [23]. A significant reduction
(P < 0.01) of phospho-(ser473)-Akt in MDA-MB-231 was observed
staining showed that compound 18 at 7 mM induced cell cycle ar-
rest at G0/G1 phase suggesting that cell cycle arrest is responsible
for cell growth inhibitory activity in MCF-7 cells also there was
increased sub-diploid population observed as compare to vehicle
control which denotes cells undergone apoptosis. A similar arrest at
G1 phase was also evident in case of DU-145 prostate cancer cells
on treatment with compound 14 (Fig. 2B).
upon treatment with 2
mM concentration for 24 h (Fig. 6A and A').
Significant decrease in phospho-Akt was also observed in DU-145
(P < 0.01) on treatment with compound 14 at 15 mM concentra-
tion (Fig. 6 B and B').
3.3. Induction of apoptosis by compound 14 and 18
4. Conclusion
Exposure of phosphatidylserine on outer leaflet of cell mem-
brane is considered as hallmark of apoptosis. Therefore, induction
of apoptosis is commonly measured in terms of binding of Annexin-
V to exposed phosphatidylserine. Annexin-V staining is further
combined with PI-staining to exclude membrane intact live cells
and necrotic cells [18]. Annexin-V and PI dual staining was carried
out and it was observed that compound 18 induced significant
apoptosis in MCF-7 cells (Fig. 3A). Annexin-V staining positive cell
were significantly increased in MCF-7 cell population treated with
compound 18. Similarly, the number of apoptotic DU-145 cells
A novel series of hybrid alkylphospholipid-DTC derivatives were
synthesized that exhibited considerable anti-cancer activity against
breast and prostate cancer. Interestingly, these compounds did not
induce any significant cytotoxicity in non-cancer cell, HEK-293. The
lead compound 18 of this series showed significant inhibition of
breast cancer cells, MCF-7 with IC50 of ~7
mM and MDA-MB-231
with IC50 of ~24 M. Compound 18 induced G0/G1 cell cycle ar-
m
rest, down-regulation of cyclins responsible for cell cycle progres-
sion (cyclin A1 and A2), induction of apoptosis and down-

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S. Jangir et al. / European Journal of Medicinal Chemistry 85 (2014) 638e647
Fig. 2. Effect of compounds 14 and 18 on cell cycle progression in MCF-7 and DU-145 cells. (A). MCF-7 cells were treated with compound 18. (B). DU-145 cells were treated with
compound 14. After 24 h, cells were harvested and stained with PI before analyzed by flow cytometry. Quantification of flow cytometry data has been shown as percent of total cells.
regulation of anti-apoptotic gene (bcl-2) and significant loss of
mitochondrial membrane potential (MMP) in MCF-7 cells. Com-
pound 18 also significantly down regulated phospho-Akt in breast
cancer cells; possibly the anti-cancer and pro-apoptotic activity of
compound 18 is due to blockade of Akt-pathway (Fig. 7).
was performed over Merck silica gel (60e120 Mesh). All chemicals
and solvents were procured from SigmaeAldrich/Merck India Ltd.
2-Bromoethyl phosphorodichloridate (3), [24] 2-bromoethyl alkyl
hydrogen phosphate (4e8) [24] and sodium salt of carbodithioic
acid (9e12) [25] were prepared by known procedure.
Similar results were observed in prostate cancer cell, DU-145
when treated with compound 14, indicating that its action is also
probably mediated through Akt like compound 18 (Fig. 7). These
new series of hybrid alkylphospholipid-DTC molecules could serve
as interesting lead against endocrine-related cancers.
5.1.1. Synthesis of 2-(hexadecyloxy(hydroxy)phosphoryloxy)ethyl
pyrrolidine-1-carbodithioate (13)
To the mixture of 2-bromoethyl hexadecyl hydrogen phosphate
(4, 1.5 g, 3.5 mmol) and methanol (20.0 mL) was added sodium
pyrrolidine-1-cabodithioate (9, 1.2 g, 7.0 mmol) and stirred at 65 ꢁC
for 3 h. The reaction mixture was concentrated under reduced
pressure. EtOAc (20.0 mL) was added into the residue, and solid salt
was filtered off. Filtrate was dried over sodium sulfate and
concentrated under reduced pressure. The crude product was pu-
rified by column chromatography over SiO2 (60e120 mesh) using
chloroform/methanol (9:1) as eluent to give the title compound
5. Experimental section
5.1. Chemistry
In general, all reagents and solvents were commercial quality
and were used without further purification. IR spectra (nmax in
cmꢀ1) of the compounds were recorded on Perkin Elmer FT-IR RX1
PC spectrophotometer. 1H NMR & 13C NMR spectra were recorded
on Bruker Supercon Magnet Avance DPX-200/DRX-300 spectrom-
eters (300 MHz for 1H; 50 and 75 MHz for 13C) and Bruker Avance III
HD-400 spectrometer (100 MHz for 13C) in deuterated solvents
(1.3 g, 75%) as light brown oil; IR (neat)
n
(cmꢀ1): 3405, 2925, 2854,
4.13e4.08 (2H, m),
1641, 1218; 1H NMR (300 MHz, CDCl3)
d
3.89e3.87 (4H, m), 3.67e3.60 (4H, m), 2.00e1.93 (4H, m), 1.60 (2H,
with TMS as internal reference (chemical shifts
d in ppm, J in Hz).
bs), 1.26 (26H, bs), 0.88 (3H, t, J ¼ 6.3 Hz); 13C NMR (50 MHz, CDCl3)
Electrospray Ionization Mass spectra (ESI-MS) were recorded on
Thermo Lcq Advantage Max-IT and HR-DART MS were recorded on
JEOL, JMS T100LC Accu TOF. Elemental analyses were performed on
Carlo Erba EA-1108 micro analyzer/Vario EL-III C H N S analyzer. All
compounds were analyzed for C, H, N and the results obtained were
within 0.4% of calculated values. The reaction progress was
routinely monitored by thin layer chromatography (TLC) on pre-
coated alumina/silica gel plates (Aldrich). Column chromatography
d 192.2 (C]S), 66.4, 64.1, 55.1, 50.7, 32.0, 30.9, 30.0, 29.8, 29.5, 26.2,
24.4, 22.8, 14.2; ESI-MS m/z 518 (MþNa); Anal. Calcd for
23H46NO4PS2: C, 55.73; H, 9.35; N, 2.83; Found: C, 55.82; H, 9.43;
N, 2.99.
The following compounds 14e32 were prepared using the
C
procedure similar to that described for compound 13 from the
appropriate 2-bromoethyl alkyl hydrogen phosphate and sodium
salt of carbodithioic acid.

S. Jangir et al. / European Journal of Medicinal Chemistry 85 (2014) 638e647
643
Fig. 3. Effect of compounds 14 and 18 on phosphatidylserine exposure on outer surface of plasma membrane in MCF-7 and DU-145 cells. (A). MCF-7 cells treated with compound 18
and (B). DU-145 cells treated with compound 14 at indicated concentration for 24 h. Cells were harvested and stained with Annexin-V and PI before analysis by flow cytometry.
Quantification of flow cytometry data has been shown as percent of total cells.
Fig. 4. Effect of compounds 14 and 18 on expression of cell cycle and apoptosis-related genes in MCF-7 and DU-145 cells. (A). MCF-7 cells treated with compound 18 at indicated
concentration for 24 h. Cells were harvested and mRNA level was determined by qPCR. (B). DU-145 cells treated with compound 14 at indicated concentration for 24 h. Cells were
harvested and Immunoblotting was performed to assess the protein expression in DU-145 cells. (B'). Bax/Bcl-2 ratio as mean SE of 3 densitometric scans, significant difference
from control is indicated as ***p < 0.001.
5.1.2. 2-(Hexadecyloxy(hydroxy)phosphoryloxy)ethyl piperidine-1-
carbodithioate (14)
J ¼ 6.2 Hz), 1.82e1.61 (8H, m), 1.25 (26H, bs), 0.88 (3H, t, J ¼ 6.6 Hz);
13C NMR (75 MHz, CDCl3)
194.8 (C]S), 65.7, 65.3, 52.7, 51.1, 37.2,
d
Eluent: 12% methanolechloroform; Yield 85%; Light yellow oil;
31.8, 29.6, 29.4, 29.2, 25.8, 22.5, 22.4, 14.0; ESI-MS m/z 532 (MþNa);
Anal. Calcd for C24H48NO4PS2: C, 56.55; H, 9.49; N, 2.75; Found: C,
56.31; H, 9.58; N, 2.82.
IR (neat)
CDCl3)
n
(cmꢀ1): 3429, 2932, 2859,1646,1219; 1H NMR (300 MHz,
d
4.33e4.03 (4H, m), 3.93e3.77 (4H, m), 3.61 (2H, t,

644
S. Jangir et al. / European Journal of Medicinal Chemistry 85 (2014) 638e647
Fig. 5. Effect of compounds 14 and 18 on MMP of MCF-7 and DU-145 cells. (A). MCF-7 cells were treated with compound 18 and (B). DU-145 cells were treated with compound 14 as
indicated concentration for 24 h. Cells were harvested, stained with JC-1 and analyzed by flow cytometry. Percent total JC-1 monomar (green) as measured by flow cytometry was
expressed as percent MMP loss. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. Effect of compounds 14 and 18 on Akt phosphorylation in MDA-MB-231 and DU-145 cells. (A). MDA-MB-231 cells were treated with compound 18 and (B). DU-145 cells were
treated with compound 14 at indicated concentration for 24 h. Cells were harvested and probed for total Akt and phospho-Akt (Ser473) by immunoblotting. (A') and (B'), histograms
showing quantitative results of western blot analysis for activation of Akt protein in MCF-7 and DU-145 cells, respectively. Significant difference from control is indicated as
**p < 0.01.
5.1.3. 2-(Hexadecyloxy(hydroxy)phosphoryloxy)ethyl azepane-1-
carbodithioate (15)
C25H50NO4PS2: C, 57.33; H, 9.62; N, 2.67; Found: C, 57.14; H, 9.48; N,
2.75.
Eluent: 10% methanolechloroform; Yield 78%; Brown oil; IR
(neat)
CDCl3)
n
(cmꢀ1): 3427, 2926, 2855, 1630, 1215; 1H NMR (300 MHz,
4.19e4.15 (2H, m), 4.06e4.04 (2H, m), 3.91e3.82 (4H, m),
5.1.4. 2-(Hexadecyloxy(hydroxy)phosphoryloxy)ethyl 4-
methylpiperazine-1-carbodithioate (16)
d
3.61e3.57 (2H, m), 1.85 (4H, bs), 1.68e1.57 (6H, m), 1.25 (26H, bs),
0.88 (3H, t, J ¼ 6.5 Hz); ESI-MS m/z 546 (MþNa); Anal. Calcd for
Eluent: 10% methanolechloroform; Yield 82%; Light yellow oil;
IR (neat) n
(cmꢀ1): 3403, 2925, 2854,1654,1218; 1H NMR (300 MHz,

S. Jangir et al. / European Journal of Medicinal Chemistry 85 (2014) 638e647
645
5.1.8. 2-(Decyloxy(hydroxy)phosphoryloxy)ethyl 4-
methylpiperazine-1-carbodithioate (20)
Eluent: 12% methanolechloroform; Yield 86%; Light yellow oil;
IR (neat)
(cmꢀ1): 3383, 2926, 2854, 1655, 1227; 1H NMR
(300 MHz, CDCl3) 4.08e4.04 (2H, m), 3.83e3.79 (2H, m),
n
d
3.63e3.59 (2H, m), 3.49 (4H, bs), 2.52e2.49 (4H, m), 2.34 (3H, s),
1.60 (2H, bs), 1.26 (14H, bs), 0.88 (3H, t, J ¼ 6.5 Hz); 13C NMR
(75 MHz, CDCl3) d 196.4 (C]S), 66.4, 64.0, 54.6, 45.7, 37.4, 32.2, 31.0,
30.9, 30.1, 30.0, 29.8, 29.6, 26.2, 22.9, 14.3; HRMS m/z calcd. for
C
C
18H37N2O4PS2 (MHþ): 441.2011; found 441.2002; Anal. Calcd for
18H37N2O4PS2: C, 49.07; H, 8.46; N, 6.36; Found: C, 49.21; H, 8.62;
N, 6.51.
5.1.9. 2-(Hydroxy(octyloxy)phosphoryloxy)ethyl pyrrolidine-1-
carbodithioate (21)
Eluent: 10% methanolechloroform; Yield 73%; yellow oil; IR
(neat)
CDCl3)
n
(cmꢀ1): 3434, 2929, 2852, 1653, 1217; 1H NMR (300 MHz,
4.08e4.02 (2H, m), 3.92e3.79 (4H, m), 3.69e3.57 (4H, m),
Fig. 7. Proposed mechanism of action of alkylphospholipid hybrids derivatives, com-
pound 14 and 18 on cancer cells.
d
2.09e1.94 (4H, m), 1.60 (2H, bs), 1.26 (10H, bs), 0.87 (3H, t,
J ¼ 6.4 Hz); 13C NMR (50 MHz, CDCl3)
d 192.3 (C]S), 66.2, 64.0, 55.2,
CDCl3)
d 4.04e4.01 (2H, m), 3.82 (7H, bs), 3.60e3.56 (1H, m), 2.51
50.8, 36.9, 32.1, 31.1, 29.8, 26.2, 24.4, 22.9, 14.3; ESI-MS m/z 406
(MþNa); Anal. Calcd for C15H30NO4PS2: C, 46.98; H, 7.88; N, 3.65;
Found: C, 47.15; H, 7.69; N, 3.74.
(4H, bs), 2.33 (3H, s), 1.59e1.57 (2H, m), 1.25 (26H, bs), 0.88 (3H, t,
J ¼ 6.6 Hz); 13C NMR (50 MHz, CDCl3)
d 196.9 (C]S), 66.4, 63.9, 54.5,
50.0, 45.6, 37.3, 32.1, 31.1, 30.1, 30.0, 29.8, 29.5, 26.2, 22.8, 14.2;
HRMS m/z calcd. for C24H49N2O4PS2 (MHþ): 525.2950; found
525.2944; Anal. Calcd for C24H49N2O4PS2: C, 54.93; H, 9.41; N, 5.34;
Found: C, 54.79; H, 9.35; N, 5.27.
5.1.10. 2-(Hydroxy(octyloxy)phosphoryloxy)ethyl piperidine-1-
carbodithioate (22)
Eluent: 8% methanolechloroform; Yield 78% Yellow oil; IR (neat)
n
d
(cmꢀ1): 3407, 2931, 2859, 1658, 1217; 1H NMR (300 MHz, CDCl3)
4.24e4.04 (3H, m), 3.84e3.80 (3H, m), 3.62e3.55 (2H, m),
5.1.5. 2-(Decyloxy(hydroxy)phosphoryloxy)ethyl pyrrolidine-1-
carbodithioate (17)
3.11e3.06 (2H, m), 1.81e1.60 (8H, m), 1.25 (10H, bs), 0.87 (3H, t,
J ¼ 6.4 Hz); 13C NMR (100 MHz, CDCl3)
d 192.0 (C]S), 65.9, 63.7,
Eluent: 8% methanolechloroform; Yield 77%; Yellow oil; IR
54.9, 50.5, 36.6, 31.5, 30.6, 30.5, 25.9, 25.4, 24.3, 24.1, 22.5, 13.9; ESI-
MS m/z 420 (MþNa); Anal. Calcd for C16H32NO4PS2: C, 48.34; H,
8.11; N, 3.52; Found: C, 48.57; H, 7.95; N, 3.68.
(neat)
CDCl3)
n
(cmꢀ1): 3395, 2927, 2857, 1654, 1219; 1H NMR (300 MHz,
4.13e4.02 (2H, m), 3.92e3.88 (2H, m), 3.84e3.79 (2H, m),
d
3.68e3.57 (4H, m), 2.12e1.92 (4H, m), 1.60 (2H, bs), 1.25 (14H, bs),
0.88 (3H, t, J ¼ 6.5 Hz); 13C NMR (75 MHz, CDCl3)
d 192.3 (C]S),
5.1.11. 2-(Hydroxy(octyloxy)phosphoryloxy)ethyl azepane-1-
carbodithioate (23)
Eluent: 12% methanolechloroform; Yield 85%; Brown oil; IR
(neat)
CDCl3)
66.1, 63.9, 55.1, 50.7, 32.0, 31.0, 30.9, 29.9, 29.8, 29.7, 29.5, 26.1, 24.5,
24.4, 22.8, 14.2; ESI-MS m/z 434 (MþNa); Anal. Calcd for
C
17H34NO4PS2: C, 49.61; H, 8.33; N, 3.40; Found: C, 49.87; H, 8.47;
n
(cmꢀ1): 3428, 2932, 2859, 1631, 1217; 1H NMR (300 MHz,
4.16 (2H, t, J ¼ 5.9 Hz), 4.06e4.02 (2H, m), 3.91e3.80 (4H,
N, 3.54.
d
m), 3.62e3.57 (2H, m), 1.85 (4H, bs), 1.57e1.56 (6H, m), 1.26 (10H,
bs), 0.89e0.85 (3H, m); 13C NMR (75 MHz, CDCl3)
d 195.9 (C]S),
5.1.6. 2-(Decyloxy(hydroxy)phosphoryloxy)ethyl piperidine-1-
carbodithioate (18)
66.1, 63.9, 55.6, 52.8, 37.3, 32.1, 29.8, 29.6, 27.5, 27.2, 26.9, 26.7, 26.4,
26.1, 22.8, 14.2; ESI-MS m/z 434 (MþNa); Anal. Calcd for
Eluent: 12% methanolechloroform; Yield 79%; Yellow oil; IR
C17H34NO4PS2: C, 49.61; H, 8.33; N, 3.40; Found: C, 49.35; H, 8.19; N,
(neat)
CDCl3)
n
(cmꢀ1): 3425, 2929, 2858, 1641, 1219; 1H NMR (300 MHz,
3.51.
d
4.34e3.97 (4H, m), 3.88e3.78 (4H, m), 3.60 (2H, t,
J ¼ 6.3 Hz), 1.81e1.59 (8H, m), 1.24 (14H, bs), 0.86 (3H, t, J ¼ 6.6 Hz);
5.1.12. 2-(Hydroxy(octyloxy)phosphoryloxy)ethyl 4-
methylpiperazine-1-carbodithioate (24)
13C NMR (75 MHz, CDCl3)
d
195.2 (C]S), 66.0, 63.8, 53.1, 51.4, 32.0,
31.0, 30.9, 29.9, 29.8, 29.7, 29.5, 26.1, 24.4, 22.8, 22.6, 14.2; ESI-MS
m/z 448 (MþNa); Anal. Calcd for C18H36NO4PS2: C, 50.80; H, 8.53;
N, 3.29; Found: C, 50.57; H, 8.61; N, 3.45.
Eluent: 10% methanolechloroform; Yield 88%; Light yellow oil;
IR (neat)
CDCl3)
3.22 (3H, bs), 2.50e2.47 (4H, m), 2.32 (3H, s), 1.61 (2H, bs), 1.26
(10H, bs), 0.89e0.85 (3H, m); 13C NMR (50 MHz, CDCl3)
196.4 (C]
n
(cmꢀ1): 3420, 2927, 2856, 1651, 1220; 1H NMR (300 MHz,
d
4.06e4.02 (3H, m), 3.85e3.79 (2H, m), 3.63e3.59 (2H, m),
d
5.1.7. 2-(Decyloxy(hydroxy)phosphoryloxy)ethyl azepane-1-
carbodithioate (19)
S), 66.2, 63.8, 54.3, 50.5, 45.5, 37.2, 32.0, 30.9, 30.8, 29.7, 29.5, 26.0,
22.7, 14.2; HRMS m/z calcd. for C16H33N2O4PS2 (MHþ): 413.1698;
found 413.1692; Anal. Calcd for C16H33N2O4PS2: C, 46.58; H, 8.06; N,
6.79; Found: C, 46.81; H, 8.23; N, 6.92.
Eluent: 8% methanolechloroform; Yield 81%; Brown oil; IR
(neat)
CDCl3)
n
(cmꢀ1): 3427, 2928, 2857, 1651, 1216; 1H NMR (300 MHz,
4.17 (2H, t, J ¼ 5.9 Hz), 4.06e4.04 (2H, m), 3.91e3.82 (4H,
d
m), 3.62e3.57 (2H, m), 1.85 (4H, bs), 1.68e1.57 (6H, m), 1.25 (14H,
5.1.13. 2-(Hexyloxy(hydroxy)phosphoryloxy)ethyl pyrrolidine-1-
carbodithioate (25)
bs), 0.87 (3H, t, J ¼ 6.6 Hz); 13C NMR (75 MHz, CDCl3)
d 195.9 (C]S),
66.1, 64.0, 55.8, 52.9, 32.1, 31.0, 29.9, 29.8, 29.7, 29.5, 27.5, 27.1, 26.8,
Eluent: 10% methanolechloroform; Yield 76%; Yellow oil; IR
26.7, 26.4, 26.1, 22.8, 14.2; ESI-MS m/z 462 (MþNa); Anal. Calcd for
(neat)
CDCl3)
n
(cmꢀ1): 3419, 2956, 2851, 1638, 1217; 1H NMR (300 MHz,
4.07e4.05 (2H, m), 3.93e3.80 (3H, m), 3.68e3.58 (3H, m),
C
19H38NO4PS2: C, 51.91; H, 8.71; N, 3.19; Found: C, 51.69; H, 8.65; N,
d
3.14.
3.21 (2H, bs), 2.09e1.95 (4H, m),1.60e1.58 (2H, m), 1.29 (6H, bs),

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S. Jangir et al. / European Journal of Medicinal Chemistry 85 (2014) 638e647
0.89e0.85 (3H, m); 13C NMR (50 MHz, CDCl3)
64.0, 55.1, 50.7, 31.8, 30.8, 26.2, 25.7, 24.6, 24.4, 22.8,14.2; ESI-MS m/
z 378 (MþNa); Anal. Calcd for C13H26NO4PS2: C, 43.93; H, 7.37; N,
3.94; Found: C, 43.79; H, 7.46; N, 3.82.
d
192.3 (C]S), 66.0,
CDCl3) d 4.19e4.04 (6H, m), 3.90e3.86 (2H, m), 3.81e3.75 (2H, m),
1.85 (4H, bs), 1.68e1.57 (6H, m), 0.91 (3H, t, J ¼ 7.3 Hz); 13C NMR
(75 MHz, CDCl3) d 195.9 (C]S), 67.3, 63.8, 55.7, 52.8, 37.3, 27.0, 26.7,
26.6, 26.3, 25.1, 10.3; ESI-MS m/z 364 (MþNa). Anal. Calcd for
C12H24NO4PS2: C, 42.21; H, 7.09; N, 4.10; Found: C, 42.37; H, 7.21; N,
5.1.14. 2-(Hexyloxy(hydroxy)phosphoryloxy)ethyl piperidine-1-
carbodithioate (26)
4.01.
Eluent: 12% methanolechloroform; Yield 82%; Light yellow oil;
5.1.20. 2-(Hydroxy(propoxy)phosphoryloxy)ethyl 4-
methylpiperazine-1-carbodithioate (32)
IR (neat)
CDCl3)
n
(cmꢀ1): 3443, 2956, 2857, 1640, 1219; 1H NMR (300 MHz,
d
4.24e4.00 (3H, m), 3.90e3.80 (3H, m), 3.63e3.59 (2H, m),
Eluent: 10% methanolechloroform; Yield 87%; Light yellow oil;
3.06e3.04 (2H, m), 1.81e1.61 (8H, m), 1.28 (6H, bs), 0.87 (3H, t,
IR (neat)
(300 MHz, CDCl3) d 4.06e4.04 (2H, m), 3.81e3.76 (6H, m), 3.61 (2H,
n
(cmꢀ1): 3449, 2958, 2809, 1654, 1221; 1H NMR
J ¼ 6.4 Hz); 13C NMR (50 MHz, CDCl3)
d
195.1 (C]S), 66.0, 63.9, 53.0,
51.6, 37.4, 31.7, 30.8, 25.6, 24.4, 22.7, 14.1; ESI-MS m/z 392 (MþNa);
Anal. Calcd for C14H28NO4PS2: C, 45.51; H, 7.64; N, 3.79; Found: C,
45.73; H, 7.49; N, 3.92.
t, J ¼ 6.1 Hz), 2.50e2.47 (4H, m), 2.32 (3H, s), 1.69e1.57 (2H, m), 0.91
(3H, t, J ¼ 7.4 Hz); 13C NMR (50 MHz, CDCl3)
d 196.5 (C]S), 67.6,
63.8, 54.4, 50.9, 45.6, 37.4, 29.7, 23.9, 10.6; HRMS m/z calcd. for
C
C
11H23N2O4PS2 (MHþ): 343.0915; found 343.0913; Anal. Calcd for
11H23N2O4PS2: C, 38.58; H, 6.77; N, 8.18; Found: C, 38.71; H, 6.85;
5.1.15. 2-(Hexyloxy(hydroxy)phosphoryloxy)ethyl azepane-1-
carbodithioate (27)
N, 8.32.
Eluent: 8% methanolechloroform; Yield 87%; Brown oil; IR
(neat)
CDCl3)
n
(cmꢀ1): 3418, 2931, 2860, 1644, 1216; 1H NMR (300 MHz,
4.18e4.04 (4H, m), 3.91e3.82 (4H, m), 3.62e3.58 (2H, m),
5.2. Biology
d
1.85 (4H, bs),1.68e1.56 (6H, m),1.28 (6H, bs), 0.87 (3H, t, J ¼ 6.4 Hz);
5.2.1. Cell culture
13C NMR (50 MHz, CDCl3)
d
195.8 (C]S), 66.1, 63.9, 55.7, 52.9, 37.4,
Human breast cancer cell lines, MCF-7 and MDA-MB-231; and
prostate cancer cell lines, PC-3 and DU-145, were originally ob-
tained from American Type of Cell Culture Collection (ATCC), USA
and stocks were maintained in laboratory. HEK-293 cells were
obtained from institutional cell repository, CSIR-CDRI. Cells were
grown in DMEM (Dulbecco modified Eagle medium, Sigma) sup-
plemented with 10% fetal bovine serum with 1ꢂ stabilized
antibiotic-antimycotic solution (Sigma) in a CO2 incubator (Sanyo,
Japan) at 37 ꢁC with 5% CO2 and 90% relative humidity [15].
31.8, 27.5, 27.1, 26.8, 26.6, 26.3, 25.7, 22.8, 14.2; ESI-MS m/z 406
(MþNa); Anal. Calcd for C15H30NO4PS2: C, 46.98; H, 7.88; N, 3.65;
Found: C, 47.16; H, 7.99; N, 3.83.
5.1.16. 2-(Hexyloxy(hydroxy)phosphoryloxy)ethyl 4-
methylpiperazine-1-carbodithioate (28)
Eluent: 10% methanolechloroform; Yield 85%; Light yellow oil;
IR(neat)
CDCl3)
n
(cmꢀ1): 3394, 2955, 2861, 1646, 1217; 1H NMR (300 MHz,
4.33e3.98 (4H, m), 3.86e3.80 (3H, m), 3.63e3.54 (3H, m),
d
2.67e2.47 (4H, m), 2.32 (3H, s), 1.60 (2H, bs), 1.29 (6H, bs), 0.88 (3H,
5.2.2. In vitro assay for anti-cancer activity
t, J ¼ 6.2 Hz); 13C NMR (75 MHz, CDCl3)
d
196.3 (C]S), 66.3, 63.8,
Anti-cancer activity of compounds was evaluated using MTT
54.4, 51.1, 49.7, 45.6, 37.2, 31.9, 30.8, 29.7, 25.7, 22.8, 14.3; HRMS m/z
calcd. for C14H29N2O4PS2 (MHþ): 385.1385; found 385.1382; Anal.
Calcd for C14H29N2O4PS2: C, 43.73; H, 7.60; N, 7.29; Found: C, 43.51;
H, 7.81; N, 7.15.
assay [16]. Briefly, 1 ꢂ 104 cells in 200
mL of DMEM, supplemented
with 10% FBS were seeded in each well of 96-well microculture
plates and incubated for 24 h at 37 ꢁC in a CO2 incubator. Com-
pounds were diluted to the desired concentrations in culture me-
dium. After 18 h of incubation, 10 mL MTT (3-(4,5-dimethylthiazol-
5.1.17. 2-(Hydroxy(propoxy)phosphoryloxy)ethyl pyrrolidine-1-
carbodithioate (29)
2-yl)-2,5-diphenyl tetrazolium bromide) (5 mg/mL) was added to
each well and the plates were further incubated for 4 h. Superna-
tant from each well was carefully removed, formazon crystals were
Eluent: 8% methanolechloroform; Yield 76%; Yellow oil; IR
(neat)
CDCl3)
n
(cmꢀ1): 3409, 2928, 2858, 1652, 1217; 1H NMR (300 MHz,
4.08e4.02 (2H, m), 3.90 (2H, t, J ¼ 6.79 Hz), 3.83e3.77 (2H,
dissolved in 100
was recorded.
mL of DMSO and absorbance at 540 nm wavelength
d
m), 3.68e3.57 (4H, m), 2.00e1.95 (4H, m),1.69e1.57 (2H, m), 0.91
(3H, t, J ¼ 7.4 Hz); 13C NMR (75 MHz, CDCl3)
d
192.0 (C]S), 67.3,
5.2.3. Cell cycle analysis
63.7, 55.0, 50.5, 36.6, 24.3, 24.1, 23.8, 10.3; ESI-MS m/z 336 (MþNa);
Anal. Calcd for C10H20NO4PS2: C, 38.33; H, 6.43; N, 4.47; Found: C,
38.17; H, 6.58; N, 4.61.
Cell cycle analysis was carried out following already reported
methods [17]. MCF-7 and DU-145 cells were plated and treated
with test compounds for 24 h, and harvested. Cells were collected,
resuspended at the rate of 1e2 ꢂ 106 cells/mL, fixed with absolute
ethanol, treated with RNase A to final concentration of 10 mg/mL,
and then stained with Propidium iodide to final concentration
5.1.18. 2-(Hydroxy(propoxy)phosphoryloxy)ethyl piperidine-1-
carbodithioate (30)
Eluent: 12% methanolechloroform; Yield 80%; Yellow oil; IR
50 mg/mL (Sigma, USA) for 30 min at room temperature. The DNA
(neat)
CDCl3)
n
(cmꢀ1): 3450, 2959, 2856, 1637, 1213; 1H NMR (300 MHz,
content of the cells was measured using a FACS Calibur flow cy-
tometer (BectoneDickinson, San Jose, CA, USA) equipped with
ModFit LT 2.0 and analyzed with Cell Quest software.
d
4.26e4.04 (4H, m), 3.91e3.78 (3H, m), 3.63e3.59 (1H, m),
3.09e3.05 (2H, m), 1.83e1.60 (8H, m), 0.92 (3H, t, J ¼ 7.3 Hz); 13C
NMR (100 MHz, CDCl3)
d 191.9 (C]S), 67.4, 63.7, 55.0, 50.6, 36.4,
25.9, 24.3, 24.1, 23.7, 10.2; ESI-MS m/z 350 (MþNa); Anal. Calcd for
5.2.4. Annexin V-PI assay for apoptosis detection
C
11H22NO4PS2: C, 40.35; H, 6.77; N, 4.28; Found: C, 40.59; H, 6.98;
Program cell death induced by the lead compound was evalu-
ated with Annexin-V-FITC/PI staining kit (SigmaeAldrich) using
flow cytometry [18]. Briefly, cells were trypsinized and washed in
PBS, 1 ꢂ 106 cells per mL were resuspended in binding buffer
(provided in the kit), and stained with FITC-conjugated annexin-V
and propidium iodide. Then, cells were incubated for 10 min in the
dark at room temperature, washed with binding buffer, and
N, 4.19.
5.1.19. 2-(Hydroxy(propoxy)phosphoryloxy)ethyl azepane-1-
carbodithioate (31)
Eluent: 10% methanolechloroform; Yield 84%; Brown oil; IR
(neat)
n
(cmꢀ1): 3402, 2929, 2853, 1622, 1218; 1H NMR (300 MHz,

S. Jangir et al. / European Journal of Medicinal Chemistry 85 (2014) 638e647
647
analyzed by flow cytometry (FACS Calibur, Becton Dickinson) and
Cell Quest software.
and K.S.S.), UGC (L.K., N.L. and V.S.), DBT (H.) and ICMR (V.B. and
V.V.) for research fellowships. This study was partially supported by
a grant from the Ministry of Health and Family Welfare, Govern-
ment of India (GAP 0001).
5.2.5. Quantitative Real Time PCR
Quantitative Real Time PCR was carried out using already pub-
lished protocol [19]. Total RNA was isolated using Trizol reagent
(Invitrogen) following the manufacturer's instructions. Total RNA
Appendix A. Supplementary data
(5 mg) was digested using DNase I (Invitrogen). The DNase-treated
Supplementary data like scanned copies of 1H NMR and 13C
NMR spectra can be found at http://dx.doi.org/10.1016/j.ejmech.
2014.08.028.
RNA was then reverse transcribed using cDNA synthesis kit (Fer-
mentas). Triplicate samples of cDNA were amplified using the Real-
Time PCR System (Roche, USA). The amplification reaction mixture
(10
m
L total volume) contained 0.5
m
L cDNA, 10
rmol forward
primer (Bcl-2: 50-agtacctgaaccggcacct-30, cyclin A1: 50-cca-
References
cagtcagggagtgcttt-30,
cyclinA2:
50-ggtactgaagtccgggaacc-30,
GAPDH: 50-gctctctgctcctcctgttc-30) and 10 pmol reverse primer
(Bcl-2: 50-ggccgtacagttccacaaa-30, cyclin A1: 50-aatgggcagta-
caggagga-30, cyclinA2: 50-gaagatccttaaggggtgca-30, GAPDH: 50-
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Acknowledgments
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We acknowledge Ms Tara Rawat (S.T.O) for technical assistance
and SAIF Division, for spectral data. We are grateful to CSIR (S.J., A.S.